The invention relates to methods of in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid or exogenous protein or peptide which are particularly suitable for use in gene therapy.
Previous studies have shown that SV40 virions disrupted at pH 10.6 [Christensen, M. and Rachmeler, M. (1976) Virology 75:433-41] or by reducing disulfide bonds [Colomar, M. C., et al. (1993) J. Virol. 67:2779-2788] may be reassociated to form infectious SV40 aggregates. The early attempts to package in vitro foreign DNA in these aggregates [Christensen and Rachmeler (1976) ibid] produced infectious products which did not resemble SV40 virions. Furthermore, their resistance to DNase has not been tested. Later, in vitro packaging experiments [Colomar et al. (1993) ibid.] did not yield particles with infectivity above the level of naked DNA.
Recently, pseudocapsids of the closely related murine polyoma virus, prepared from polyoma VP1, were used as carriers for heterologous DNA into mammalian cells [Forstova, J., et al. (1995) Hum. Gene Therapy 6:297-306]. The pseudo-capsid protected 2-30% of the input DNA from DNase I digestion. When a plasmid carrying the cat gene was tested, most of the DNA which was protected from DNase I appeared as a xcx9c2 kb fragment, while the input plasmid was significantly larger (exact size was not reported), suggesting that each DNA molecule was only partially protected against DNases. Infectious units were not measured in those experiments. The DNA transferred into recipient cells was functional in gene expression, albeit at a very low efficiency. With a 1.6 kb DNA fragment which carries the polyoma middle T-antigen,  less than 30 transformed foci were obtained per 1 xcexcg of input DNA. Similarly, a low level of CAT activity was observed with the plasmid carrying the cat gene.
SV40 is a simian papovirus, with a small double-stranded circular DNA genome of 5.2 kb [reviewed in Tooze, J. (1981) DNA Tumor Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.]. The viral capsid, surrounding the viral mini-chromosome, is composed of three viral-coded proteins, VP1, VP2, and VP3. Recent X-ray crystallographic studies on SV40 structure at 3.8 xc3x85 resolution [Liddington, R., et al. (1991) Nature 354:278-282] revealed that the outer shell of the virion particle is composed of 72 pentamers of VP1, 60 hexavalent and 12 pentavalent. The VP2 and VP3 appear to bridge between the VP1 outer shell and the chromatin core. The VP1 pentamers have identical conformations, except for the carboxy-terminal arms, which tie them together. Five arms extend from each pentamer and insert into the neighboring pentamers in three distinct kinds of interactions. It appears that this construction facilitates the use of identical building blocks in the formation of a structure that is sufficiently flexible as required for the variability in packing geometry [Liddington et al. (1991) ibid.].
Another protein encoded by the late regions of SV40 (which also encoded the three capsid proteins VP1, VP2 and VP3) is the agnoprotein, also called LP1. This is protein a small, 61 amino acid protein. Although the agnoprotein was not found in the viral capsid, it is thought to expedite viral assembly in vivo [Resnick, J and Shenk, T. (1986) J. Virol. 60:1098-1106; Ng, S. C., et al. (1985) J. Biol. Chem. 260:1127-1132; Carswell, S. and Alwine, J. C. (1986) J. Virol. 60:1055-1061].
The major hindrance in beginning to use the SV40 pseudovirions in preliminary experiments in humans is the present need for a viral helper for encapsidation. This results in pseudoviral stocks that contain also wild type SV40. Because of the similarity in properties (shape, size and density) between the pseudovirions and the helper, they cannot be separated by physical means. An ideal way to prepare pseudovirions for therapeutic purposes for human use would be by in vitro packaging. This would provide maximal safety, since all steps of the preparation can be well controlled. Ex vivo administration would circumvent problems associated with immune response.
Viral packaging in vivo occurs by gradual addition and organization of capsid proteins around the SV40 chromatin [Garber, E. A., et al. (1980) Virology 107: 389-401; Bina, M. (1986) Comments Mol. Cell Biophys. 4:55]. The three capsid proteins VP1, VP2 and VP3 bind to DNA non-specifically [Soussi, T. (1986) J. Virol. 59:740-742; Clever, J., et al. (1993) J. Biol. Chem. 268:20877-20883]. How the specific recognition between the viral capsid proteins and its DNA is achieved remains unclear. The packaging of SV40 using pseudovirions, in which most of the viral DNA is replaced by other sequences has been investigated [Oppenheim, A., et al. (1986) Proc. Natl. Acad. Sci. USA 83:6925-6929]. The pseudoviral particles are prepared by encapsidating plasmids that carry the SV40 origin of replication (ori) and the packaging signal (ses) [Oppenheim, A., et al. (1992) J. Virol. 66:5320-5328]. The model suggests that ses serves several functions in SV40 packaging: as a sensor for the level of the late viral proteins in the transition from replication and/or transcription to packaging, in nucleosomal reorganization and the initiation of viral assembly [Oppenheim, A., et al. (1994). J. Mol. Biol. 238:501-513] and probably also as a nucleation center for viral assembly [Dalyot-Herman, N. et al. (1996) J. Mol. Biol. 259:69-80].
The pseudovirions, carrying various genes of therapeutic interest, are very efficient in DNA transfer into a wide range of cells, including human bone marrow cells, and are therefore potential vectors for gene therapy [Oppenheim et al. (1986) ibid.; Oppenheim A., et al. (1987) Ann. New York Acad. Sci. 511:418-427; Dalyot, N. and Oppenheim, A. (1989) Efficient transfer of the complete human beta-globin gene into human and mouse hemopoietic cells via SV40 pseudovirions. In: Gene Transfer and Gene Therapy (Beaudet, A. L., Mulligan R, I. M. Verma, eds), pp. 47-56, Alan R. Liss, Inc., New York; Oppenheim, A., et al. (1992) Development of somatic gene therapy: A simian virus 40 pseudoviral vector for hemopoietic cells. In Genetic Among Jews (Bonne-Tamir, B., A. Adams, eds), pp. 365-373, Oxford University Press, Oxford]. The ideal way to prepare pseudovirions for therapeutic purposes for human use is by in vitro packaging. This would provide maximal safety, since all steps of the preparation can be well controlled.
The present invention relates to construct capable of infecting a mammalian cell comprising at least one semi-purified or pure SV40 capsid protein and a constituent selected from the group consisting of an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, a vector comprising an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product, a vector comprising an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product, an exogenous protein or peptide product, and antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in said mammalian cell; and optionally further comprising operatively linked regulatory elements sufficient for the expression and/or replication of said exogenous protein in a mammalian cell.
The construct of the invention may optionally further comprise additional SV40 protein or proteins, preferably SV40 agnoprotein.
Constructs according to the invention may comprise as said constituent exogenous circular or linear DNA encoding an exogenous protein or peptide product, or is itself a therapeutic product, or a vector comprising exogenous DNA encoding a therapeutic RNA, or encoding an exogenous protein or peptide product.
The said protein product is preferably a therapeutic protein or peptide product which is not made or contained in mammalian cells, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in abnormally low amount, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in defective form or is DNA which encodes a therapeutic protein or peptide product which is made or contained in mammalian cells in physiologically abnormal or normal amount and can be an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.
The constructs of the invention may comprise SV40-derived ori DNA sequence as said replication regulatory element and may further comprise DNA sequences encoding one or more regulatory elements sufficient for the expression of said exogenous RNA or exogenous protein or peptide in said mammalian cell.
In other embodiments, in constructs according to the invention said constituent is exogenous RNA, preferably RNA which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made contained in said cell in defective form, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount, said RNA having regulatory elements, including translation signal/s sufficient for the translation of said protein or peptide product in said mammalian cell, operatively linked thereto.
In other embodiments, the constructs according to the invention may comprise as said constituent an exogenous protein or peptide product, which can be a therapeutic protein or peptide product which is not made or contained in mammalian cells, or is a therapeutic protein or peptide product which is made or contained in such cells in abnormally low amount, or is a therapeutic protein or peptide product which is made or contained in such cells in defective form or is a therapeutic protein or peptide product which is made or contained in mammalian cells in physiologically abnormal or normal amount.
In further embodiments, the constructs according to the invention may comprise as said constituent antisense RNA or DNA or ribozyme RNA, or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in mammalian cells.
The mammalian cells can be hemopoietic cells, such as bone marrow cells, peripheral blood cells and cord blood cells or liver cells, epithelial cells, endothelial cells, liver cells, epidermal cells, muscle cells, tumor cells, nerve cells and germ line cells.
The invention further relates to a method for the in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid comprising the steps of bringing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins into contact with the exogenous nucleic acid to give recombinant SV40 viruses or with a vector comprising the exogenous nucleic acid to give pseudoviruses; and optionally subjecting the SV40 viruses or pseudo-viruses thus formed to digestion by nuclease to remove non-packaged DNA.
In the method of the invention, at least one other SV40 protein, preferably SV40 agnoprotein, can be added to the mixture of the SV40 capsid protein/s and the nucleic acid.
The DNA employed in the method of the invention can be DNA which encodes a therapeutic protein or peptide product which is not made or contained in mammalian cells, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in abnormally low amount, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in defective form or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in physiologically abnormal or normal amount or is DNA which encodes a therapeutic RNA.
The therapeutic protein or peptide product can be an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.
The nucleic acid employed in the method of the invention can alternatively be exogenous RNA, wherein said RNA is RNA which encodes a therapeutic protein or peptide product which is not made or contained in mammalian cells, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in defective form or is RNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in physiologically abnormal or normal amount and wherein said RNA has regulatory elements, including translation signal, sufficient for the translation of said protein product in mammalian cells, operatively linked thereto.
The method of the invention can also be used for the in vitro construction of recombinant SV40 viruses or pseudoviruses comprising an exogenous protein or peptide comprising the steps of bringing a semi-purified or purified SV40 capsid protein or a mixture of at least two such proteins into contact with the exogenous protein to give recombinant SV40 viruses or pseudoviruses; and optionally purifying the recombinant viruses or pseudoviruses thus obtained from any non-packaged protein.
In addition, the method of the invention can be used for the in vitro construction of SV40 pseudoviruses comprising exogenous antisense RNA, or ribozyme RNA or RNA or DNA which inhibits or prevents the expression of undesired protein/s in a mammalian cell, comprising the steps of bringing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins into contact with the exogenous antisense RNA, or ribozyme RNA, or RNA or DNA which inhibits or prevents the expression of undesired protein/s in a mammalian cell, to give recombinant SV40 pseudoviruses; and optionally subjecting the SV40 pseudoviruses thus formed to digestion by nuclease to remove non-packaged DNA.
In a further aspect, the invention relates to mammalian cells infected with the constructs of the invention or with constructs obtained by any of the methods of the invention.
Still further, the invention relates to a method of providing a therapeutic DNA, RNA, protein or peptide product or antisense RNA to a patient in need of such product by administering to the patient a therapeutically effective amount of the SV40 viruses or pseudoviruses of the invention or a therapeutically effective amount of infected cells according to the invention.
Pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the SV40 viruses or pseudoviruses according to the invention or a therapeutically effective amount of infected cells according to the invention are also within scope of this application.